Nico Peiren

Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage.

Honey bee workers use venom for the defence of the colony and themselves when they are exposed to dangers and predators. It is produced by a long thin, convoluted, and bifurcated gland, and consists of several toxic proteins and peptides. The present study was undertaken in order to identify the mechanisms that protect the venom gland secretory cells against these harmful components. Samples of whole venom glands, including the interconnected reservoirs, were separated by two-dimensional gel electrophoresis and the most abundant protein spots were subjected to mass spectrometric identification using MALDI TOF/TOF-MS and LC MS/MS. This proteomic study revealed four antioxidant enzymes: CuZn superoxide dismutase (SOD1), glutathione-S-transferase sigma 1 isoform A (GSTS1), peroxiredoxin 2540 (PXR2540) and thioredoxin peroxidase 1 isoform A (TPX1). Although glutathione-S-transferase (GST) has also been associated with xenobiotic detoxification, the protein we found belongs to the GST Sigma class which is known to protect against oxidative stress only. Moreover, we could demonstrate that the GST and SOD activity of the venom gland was low and moderate, respectively, when compared to other tissues from the adult honey bee. Several proteins involved in other forms of stress were likewise found but it remains uncertain what their function is in the venom gland. In addition to major royal jelly protein 9 (MRJP9), already found in a previous proteomic study, we identified MRJP8 as second member of the MRJP protein family to be associated with the venom gland. Transcripts of both MRJPs were amplified and sequenced. Two endocuticular structural proteins were abundantly present in the 2D-gel and most probably represent a structural component of the epicuticular lining that protects the secretory cells from the toxins they produce.

Molecular cloning and expression of icarapin, a novel IgE-binding bee venom protein

The 1045 bp full‐length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672 bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N‐[TS]‐T‐S‐[TV]‐x‐K‐[VI](2)‐[DN]‐G‐H‐x‐V‐x‐I‐N‐[ED]‐T‐x‐Y‐x‐[DHK]‐x(2,6)‐ [STA]‐[VLFI]‐x‐[KR]‐V‐R‐[VLI]‐[IV]‐[DN]‐V‐x‐P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct.

Two novel proteins expressed by the venom glands of Apis mellifera and Nasonia vitripennis share an ancient C1q-like domain.

An in‐depth proteomic study of previously unidentified two‐dimensional polyacrylamide gel electrophoresis spots of honey bee (Apis mellifera, Hymenoptera) venom revealed a new protein with a C1q conserved domain (C1q‐VP). BlastP searching revealed a strong identity with only two proteins from other insect species: the jewel wasp, Nasonia vitripennis (Hymenoptera), and the green pea aphid, Acyrthosiphon pisum (Hemiptera). In higher organisms, C1q is the first subcomponent of the classical complement pathway and constitutes a major link between innate and acquired immunity. Expression of C1q‐VP in a variety of tissues of honey bee workers and drones was demonstrated. In addition, a wide spatial and temporal pattern of expression was observed in N. vitripennis. We suggest that C1q‐VP represents a new member of the emerging group of venom trace elements. Using degenerate primers the corresponding gene was found to be highly conserved in eight hymenopteran species, including species of the Aculeata and the Parasitica groups (suborder Apocrita) and even the suborder Symphyta. A preliminary test using recombinant proteins failed to demonstrate Am_C1q‐VP‐specific immunoglobulin E recognition by serum from patients with a documented severe bee venom allergy.

Genomic and transcriptional analysis of protein heterogeneity of the honeybee venom allergen Api m 6

Several components of honeybee venom are known to cause allergenic responses in humans and other vertebrates. One such component, the minor allergen Api m 6, has been known to show amino acid variation but the genetic mechanism for this variation is unknown. Here we show that Api m 6 is derived from a single locus, and that substantial protein‐level variation has a simple genome‐level cause, without the need to invoke multiple loci or alternatively spliced exons. Api m 6 sits near a misassembled section of the honeybee genome sequence, and we propose that a substantial number of indels at and near Api m 6 might be the root cause of this misassembly. We suggest that genes such as Api m 6 with coding‐region or untranslated region indels might have had a strong effect on the assembly of this draft of the honeybee genome.

Feeding strategy of two sympatric anostracan species (Crustacea)

The transport rate of chalk, clay particles and algal cells (Scenedesmus sp.) through the digestive tract of Streptocephalus torvicornis and Branchipus schaefferi is described under experimental conditions. Differences in transport rate as well as in the degree of digestion at a fixed particle density of algae were found. In S. torvicornis, the transport rate is higher and the digestion of algae lower than in B. schaefferi. These differences might reflect trophic differences related to niche partitioning in these sympatric species.